Ubiquitin is associated with early truncation of tau protein at aspartic acid(421) during the maturation of neurofibrillary tangles in Alzheimer's disease

Abstract

Pathological processing of tau protein during the formation and maturation of neurofibrillary tangles (NFTs) includes abnormal phosphorylation, conformational changes and truncation of the C-terminus at aspartic-acid(421) (apoptotic product) and glutamic-acid(391) residues. Abnormal phosphorylation and misfolding may serve as recognition signals for ubiquitin-targeting and proteosomal processing. For this reason, we sought to determine whether ubiquitin-targeting of tau is associated with particular tau modifications that herald specific stages of NFTs maturation in the hippocampus of Alzheimer's disease cases. Using multiple tau antibodies, we found that 30% of the total load of NFTs is ubiquitin-associated. As reported previously ubiquitin immunoreactivity was associated with markers of phosphorylated tau in certain NFTs; however, a strong association was also found between ubiquitin and the earliest known truncation event at aspartic-acid(421) . These findings indicate that tau protein in the NFTs may be dually subjected to both apoptotic and proteosomal processing. By contrast ubiquitin immunoreactivity was poorly associated with truncation of tau at glutamic-acid(391) , suggesting that this proteolytic event may be independent of proteosomal activity. It would appear, therefore, that ubiquitin targeting of tau protein occurs at NFTs in the early and intermediate stages of the maturation.

Figure 1

Ubiquitin is associated with tau protein in neurofibrillary tangles (NFTs). Ubiquitin‐positive NFTs (arrows) and clusters of neuropil threads are observed by immunoperoxidase in the hippocampus of Alzheimer's disease (AD) patients (A). Confocal microscopy images of ubiquitin immunoreactivity and thiazin red (TR) staining (B). Note in the merge channel, the colocalization displayed in some NFTs (arrows). However ubiquitin (Ubi) is not present in all the NFTs detected by TR (asterisk). Dot‐blot analysis of the A68 fraction, purified from the brain of two AD patients, also confirms that ubiquitin is a constituent of fibrillary aggregates of tau protein (C). Positive spots correspond to the use of a polyclonal antibody to ubiquitin, and two monoclonal antibodies to tau protein (Tau‐46.1 and Tau‐5). Scale bars 35 µm in (A), and 6 µm in panel (B).

Figure 2

Colocalization between ubiquitin and tau protein in the neurofibrillary tangles (NFTs) of demented patients. Double‐labeling immunofluorescence and confocal microscopy illustrate the patterns of colocalization between ubiquitin (ub: polyclonal antibody to ubiquitin) and different presentations of tau protein (red channel) in the NFTs. Those structures composed of Asp⁴²¹‐truncated tau (Tau‐C3 antibody), Ser^396,404‐phosphorylated tau [paired helical filaments (PHF)1 antibody] and C‐terminus intact tau protein (Tau‐46.1 antibody) were constantly attached by ubiquitin [see arrows in the merge channels of (A–C)]. However, almost no colocalization was found between ubiquitin and the Glu³⁹¹‐truncated tau protein (MN423 antibody) in most of the NFTs analyzed (D). Note in (D) that the NFTs recognized by either ubiquitin or MN423 have a different morphology. Moreover ubiquitin was commonly detected in neuropil threads and dystrophic neurites in addition to the NFTs. In (D), lpfc: lipofucsin. Scale bars 27 µm in (A), 10 µm in (B), 6 µm in (C) and 14 µm in (D).

Figure 3

Quantitative colocalization of ubiquitin and tau proteins in neurofibrillary tangles (NFTs). A. NFTs immunoreactive to distinct antibodies to tau protein. A significant difference was found by using a one‐way analysis of variance (ANOVA) (P = 0.009) followed by Tukey's multiple comparison test to determine specific differences between all the tau antibodies. Only the number of NFTs immunoreactive to paired helical filaments (PHF)1 was significantly greater than that of the NFTs recognized by the Tau‐46.1 antibody. B. In the whole group of demented patients, colocalization was expressed as the percentage of NFTs composed of ubiquitin (Ub: polyclonal antibody to ubiquitin) and tau protein (recognized independently by Tau‐C3, MN423, PHF1 and Tau‐46.1). Colocalization was found in most of the combinations; however, almost undetected numbers of NFTs were composed of ubiquitin and tau truncated at the Glu³⁹¹ (Ub ‐MN423), which was also consistent when the group was separated as AD (C) and mixed dementia patients (D). The same statistical analysis as in (A) was done in (B–D) (see the text for details). *P < 0.05; **P < 0.001.

Figure 4

In neurofibrillary tangles (NFTs), ubiquitin colocalizes with Asp⁴²¹‐truncated tau but not with tau cleaved at the Glu³⁹¹. Triple labeling by including the Alz‐50 antibody confirms that early NFTs detected by the Tau‐C3 antibody (for Asp⁴²¹‐truncated tau) are also composed of ubiquitin (ub) (A) and (B). In contrast, mature NFTs immunoreactive to MN423 (to tau cleaved at the Glu³⁹¹) were little composed of ubiquitin (C–D). Note in (B) the coexistence for the three markers in the same NFT, whereas in (C) only the double‐labeled NFTs are composed of ubiquitin and stained with Alz‐50 antibody. In panels (C–D), the NFTs recognized by MN423 remain unassociated with other markers and have an unmerged blue color (see the merge channel). These MN423‐positive NFTs sometimes were surrounded by a crown of neuropil treads composed of ubiquitin [see merge channel in (D)]. Scale bars 9 µm in (A), 10 µm in (B), 22 µm in (C) and 24 µm in (D).

Figure 5

Ubiquitin targeting is associated with Asp⁴²¹‐truncation but not with Glu³⁹¹‐cleavage of tau during the maturation of the NFTs in AD. At early stages of the maturation of the NFTs, the major modification in tau is the abnormal phosphorylation of several residues (A–B). Truncation of tau at Asp⁴²¹ may also occur at early stages, but it is more predominant at intermediate stages (C–D). In late‐stage NFTs (E), Glu³⁹¹‐truncated tau remains as the major component. Ubiquitin targeting of tau seems to occur at early and mostly at intermediate stages of the maturation of the NFTs (B–D), in close association with abnormal phosphorylation and Asp⁴²¹‐truncation (B–D). Scant association is found between ubiquitin and Glu³⁹¹‐truncated tau (E).